The present invention relates generally to the fabrication of an integrated miniaturized fluidic system fabricated using Micro-ElectroMechanical System (MEMS) technology, particularly to the improved fabrication of a microsized electrospray nozzle.
Microchip-based separation devices have been developed for rapid analysis of large numbers of samples. Compared to other conventional separation devices, these microchip-based separation devices have higher sample throughput, reduced sample and reagent consumption, and reduced chemical waste. The liquid flow rates for microchip-based separation devices range from approximately 1-300 nanoliters per minute for most applications. Examples of microchip-based separation devices include those for capillary electrophoresis (xe2x80x9cCExe2x80x9d), capillary electrochromatography (xe2x80x9cCECxe2x80x9d) and high-performance liquid chromatography (xe2x80x9cHPLCxe2x80x9d) include Harrison et al., Science 261:859-97 (1993); Jacobson et al., Anal. Chem. 66:1114-18 (1994), Jacobson et al., Anal. Chem. 66:2369-73 (1994), Kutter et al., Anal. Chem. 69:5165-71 (1997) and He et al., Anal. Chem. 70:3790-97 (1998). Such separation devices are capable of fast analyses and provide improved precision and reliability compared to other conventional analytical instruments.
The work of He et al., Anal. Chem. 70:3790-97 (1998) demonstrates some of the types of structures that can be fabricated in a glass substrate. This work shows that co-located monolithic support structures (or posts) can be etched reproducibly in a glass substrate using reactive ion etching (RIE) techniques. Currently, anisotropic RIE techniques for glass substrates are limited to etching features that are 20 xcexcm or less in depth. This work shows rectangular 5 xcexcm by 5 xcexcm width by 10 xcexcm in depth posts and stated that deeper structures were difficult to achieve. The posts are also separated by 1.5 xcexcm. The posts supports the stationary phase just as with the particles in LC and CEC columns. An advantage to the posts over conventional LC and CEC is that the stationary phase support structures are monolithic with the substrate and therefore, immobile.
He et. al., also describes the importance of maintaining a constant cross-sectional area across the entire length of the separation channel. Large variations in the cross-sectional area can create pressure drops in pressure driven flow systems. In electrokinetically driven flow systems, large variations in the cross-sectional area along the length of a separation channel can create flow restrictions that result in bubble formation in the separation channel. Since the fluid flowing through the separation channel functions as the source and carrier of the mobile solvated ions, formation of a bubble in a separation channel will result in the disruption of the electroosmotic flow.
Electrospray ionization provides for the atmospheric pressure ionization of a liquid sample. The electrospray process creates highly-charged droplets that, under evaporation, create ions representative of the species contained in the solution. An ion-sampling orifice of a mass spectrometer may be used to sample these gas phase ions for mass analysis. When a positive voltage is applied to the tip of the capillary relative to an extracting electrode, such as one provided at the ion-sampling orifice of a mass spectrometer, the electric field causes positively-charged ions in the fluid to migrate to the surface of the fluid at the tip of the capillary. When a negative voltage is applied to the tip of the capillary relative to an extracting electrode, such as one provided at the ion-sampling orifice to the mass spectrometer, the electric field causes negatively-charged ions in the fluid to migrate to the surface of the fluid at the tip of the capillary.
When the repulsion force of the solvated ions exceeds the surface tension of the fluid being electrosprayed, a volume of the fluid is pulled into the shape of a cone, known as a Taylor cone, which extends from the tip of the capillary. A liquid jet extends from the tip of the Taylor cone and becomes unstable and generates charged-droplets. These small charged droplets are drawn toward the extracting electrode. The small droplets are highly-charged and solvent evaporation from the droplets results in the excess charge in the droplet residing on the analyte molecules in the electrosprayed fluid. The charged molecules or ions are drawn through the ion-sampling orifice of the mass spectrometer for mass analysis. This phenomenon has been described, for example, by Dole et al., Chem. Phys. 49:2240 (1968) and Yamashita et al., J. Phys. Chem. 88:4451 (1984). The potential voltage (xe2x80x9cVxe2x80x9d) required to initiate an electrospray is dependent on the surface tension of the solution as described by, for example, Smith, IEEE Trans. Ind. Appl. 1986, IA-22:527-35 (1986). Typically, the electric field is on the order of approximately 106 V/m. The physical size of the capillary and the fluid surface tension determines the density of electric field lines necessary to initiate electrospray.
When the repulsion force of the solvated ions is not sufficient to overcome the surface tension of the fluid exiting the tip of the capillary, large poorly charged droplets are formed. Fluid droplets are produced when the electrical potential difference applied between a conductive or partly conductive fluid exiting a capillary and an electrode is not sufficient to overcome the fluid surface tension to form a Taylor cone.
Electrospray Ionization Mass Spectrometry: Fundamentals, Instrumentation, and Applications, edited by R. B. Cole, ISBN 0-471-14564-5, John Wiley and Sons, Inc., New York summarizes much of the fundamental studies of electrospray. Several mathematical models have been generated to explain the principals governing electrospray. Equation 1 defines the electric field Ec at the tip of a capillary of radius rc with an applied voltage Vc at a distance d from a counter electrode held at ground potential:                               E          c                =                              2            ⁢                          V              c                                                          r              c                        ⁢                          ln              ⁡                              (                                  4                  ⁢                                      d                    /                                          r                      c                                                                      )                                                                        (        1        )            
The electric field Eon required for the formation of a Taylor cone and liquid jet of a fluid flowing to the tip of this capillary is approximated as:                               E          on                ≈                              (                                          2                ⁢                γcosθ                                                              ϵ                  o                                ⁢                                  r                  c                                                      )                                1            /            2                                              (        2        )            
where xcex3 is the surface tension of the fluid, xcex8 is the half-angle of the Taylor cone and xcex50 is the permittivity of vacuum. Equation 3 is derived by combining equations 1 and 2 and approximates the onset voltage Von required to initiate an electrospray of a fluid from a capillary:                               V          on                ≈                                            (                                                                    r                    c                                    ⁢                  γcosθ                                                  2                  ⁢                                      ϵ                    0                                                              )                                      1              /              2                                ⁢                      ln            ⁡                          (                              4                ⁢                                  d                  /                                      r                    c                                                              )                                                          (        3        )            
As can be seen by examination of equation 3, the required onset voltage is more dependent on the capillary radius than the distance from the counter-electrode.
It would be desirable to define an electrospray device that could form a stable electrospray of all fluids commonly used in CE, CEC, and LC. The surface tension of solvents commonly used as the mobile phase for these separations range from 100% aqueous (xcex3=0.073 N/m) to 100% methanol (xcex3=0.0226 N/m). As the surface tension of the electrospray fluid increases, a higher onset voltage is required to initiate an electrospray for a fixed capillary diameter. As an example, a capillary with a tip diameter of 14 xcexcm is required to electrospray 100% aqueous solutions with an onset voltage of 1000 V. The work of M. S. Wilm et al., Int. J. Mass Spectrom. Ion Processes 136:167-80 (1994), first demonstrates nanoelectrospray from a fused-silica capillary pulled to an outer diameter of 5 xcexcm at a flow rate of 25 nL/min. Specifically, a nanoelectrospray at 25 nL/min was achieved from a 2 xcexcm inner diameter and 5 xcexcm outer diameter pulled fused-silica capillary with 600-700 V at a distance of 1-2 mm from the ion-sampling orifice of an electrospray equipped mass spectrometer.
Electrospray in front of an ion-sampling orifice of an API mass spectrometer produces a quantitative response from the mass spectrometer detector due to the analyte molecules present in the liquid flowing from the capillary. One advantage of electrospray is that the response for an analyte measured by the mass spectrometer detector is dependent on the concentration of the analyte in the fluid and independent of the fluid flow rate. The response of an analyte in solution at a given concentration would be comparable using electrospray combined with mass spectrometry at a flow rate of 100 xcexcL/min compared to a flow rate of 100 nL/min. D. C. Gale et al., Rapid Commun. Mass Spectrom. 7:1017 (1993) demonstrate that higher electrospray sensitivity is achieved at lower flow rates due to increased analyte ionization efficiency. Thus by performing electrospray on a fluid at flow rates in the nanoliter per minute range provides the best sensitivity for an analyte contained within the fluid when combined with mass spectrometry.
Thus, it is desirable to provide an electrospray device for integration of microchip-based separation devices with API-MS instruments. This integration places a restriction on the capillary tip defining a nozzle on a microchip. This nozzle will, in all embodiments, exist in a planar or near planar geometry with respect to the substrate defining the separation device and/or the electrospray device. When this co-planar or near planar geometry exists, the electric field lines emanating from the tip of the nozzle will not be enhanced if the electric field around the nozzle is not defined and controlled and, therefore, an electrospray is only achievable with the application of relatively high voltages applied to the fluid.
Attempts have been made to manufacture an electrospray device for microchip-based separations. Ramsey et al., Anal. Chem. 69:1174-78 (1997) describes a microchip-based separations device coupled with an electrospray mass spectrometer. Previous work from this research group including Jacobson et al., Anal. Chem. 66:1114-18 (1994) and Jacobson et al., Anal. Chem. 66:2369-73 (1994) demonstrate impressive separations using on-chip fluorescence detection. This more recent work demonstrates nanoelectrospray at 90 nL/min from the edge of a planar glass microchip. The microchip-based separation channel has dimensions of 10 xcexcm deep, 60 xcexcm wide, and 33 mm in length. Electroosmotic flow is used to generate fluid flow at 90 nL/min. Application of 4,800 V to the fluid exiting the separation channel on the edge of the microchip at a distance of 3-5 mm from the ion-sampling orifice of an API mass spectrometer generates an electrospray. Approximately 12 nL of the sample fluid collects at the edge of the microchip before the formation of a Taylor cone and stable nanoelectrospray from the edge of the microchip. The volume of this microchip-based separation channel is 19.8 nL. Nanoelectrospray from the edge of this microchip device after capillary electrophoresis or capillary electrochromatography separation is rendered impractical since this system has a dead-volume approaching 60% of the column (channel) volume. Furthermore, because this device provides a flat surface, and, thus, a relatively small amount of physical asperity for the formation of the electrospray, the device requires an impractically high voltage to overcome the fluid surface tension to initiate an electrospray.
Xue, Q. et al., Anal. Chem. 69:426-30 (1997) also describes a stable nanoelectrospray from the edge of a planar glass microchip with a closed channel 25 xcexcm deep, 60 xcexcm wide, and 35-50 mm in length. An electrospray is formed by applying 4,200 V to the fluid exiting the separation channel on the edge of the microchip at a distance of 3-8 mm from the ion-sampling orifice of an API mass spectrometer. A syringe pump is utilized to deliver the sample fluid to the glass microchip at a flow rate of 100 to 200 nL/min. The edge of the glass microchip is treated with a hydrophobic coating to alleviate some of the difficulties associated with nanoelectrospray from a flat surface that slightly improves the stability of the nanoelectrospray. Nevertheless, the volume of the Taylor cone on the edge of the microchip is too large relative to the volume of the separation channel, making this method of electrospray directly from the edge of a microchip impracticable when combined with a chromatographic separation device.
T. D. Lee et. al., 1997 International Conference on Solid-State Sensors and Actuators Chicago, pp. 927-30 (Jun. 16-19, 1997) describes a multi-step process to generate a nozzle on the edge of a silicon microchip 1-3 xcexcm in diameter or width and 40 xcexcm in length and applying 4,000 V to the entire microchip at a distance of 0.25-0.4 mm from the ion-sampling orifice of an API mass spectrometer. Because a relatively high voltage is required to form an electrospray with the nozzle positioned in very close proximity to the mass spectrometer ion-sampling orifice, this device produces an inefficient electrospray that does not allow for sufficient droplet evaporation before the ions enter the orifice. The extension of the nozzle from the edge of the microchip also exposes the nozzle to accidental breakage. More recently, T. D. Lee et. al., in 1999 Twelfth IEEE International Micro Electro Mechanical Systems Conference (Jan. 17-21, 1999), presented this same concept where the electrospray component was fabricated to extend 2.5 mm beyond the edge of the microchip to overcome this phenomenon of poor electric field control within the proximity of a surface.
Thus, it is also desirable to provide an electrospray device with controllable spraying and a method for producing such a device that is easily reproducible and manufacturable in high volumes.
U.S. Pat. No. 5,501,893 to Laermer et. al., reports a method of anisotropic plasma etching of silicon (Bosch process) that provides a method of producing deep vertical structures that is easily reproducible and controllable. This method of anisotropic plasma etching of silicon incorporates a two step process. Step one is an anisotropic etch step using a reactive ion etching (RIE) gas plasma of sulfur hexafluoride (SF6). Step two is a passivation step that deposits a polymer on the vertical surfaces of the silicon substrate. This polymerizing step provides an etch stop on the vertical surface that was exposed in step one. This two step cycle of etch and passivation is repeated until the depth of the desired structure is achieved. This method of anisotropic plasma etching provides etch rates over 3 xcexcm/min of silicon depending on the size of the feature being etched. The process also provides selectivity to etching silicon versus silicon dioxide or resist of greater than 100:1 which is important when deep silicon structures are desired. Laermer et. al., in 1999 Twelfth IEEE International Micro Electro Mechanical Systems Conference (Jan. 17-21, 1999), reported improvements to the Bosch process. These improvements include silicon etch rates approaching 10 xcexcm/min, selectivity exceeding 300:1 to silicon dioxide masks, and more uniform etch rates for features that vary in size.
The area of miniaturized microfluidic technology, also known as xe2x80x9clab-on-a-chipxe2x80x9d or xe2x80x9cmicro total analysis systemsxe2x80x9d is growing fast with the promise of revolutionizing chemical analysis and offering enabling tools and technologies for the life sciences. The majority of interesting molecules and compounds in the life sciences are in the liquid phase and typically analytical measurements are used to conduct quantitative and qualitative trace analysis of these analytes in solution. The need for the ability to test low sample amounts and volumes is constantly increasing as seen in drug discovery and drug development, including pharmacokinetic and proteomics applications, pushing the need for better analytical measurements. Inherently, microfluidics are a xe2x80x9cgood fitxe2x80x9d for the move to both smaller sample and volume requirements. In fact, the main reason for miniaturization has been to enhance analytical performance of the device rather than reduce its over physical size. Additionally, the miniaturizing analysis with microfluidics allows for integration of multiple separation techniques that enable parallel processing and also for the incorporation of several types of analytical measurements in a single device (sample handling, injections, 2D separations, reaction chambers etc.). Inherently, there are other benefits with the miniaturization such as reduction in reagent and waste disposal as well as the device footprint.
Inherent to microfabrication is alignment of photolithography masks for pattern transfer to the substrate (wafer). For more complex devices, such as electrospray nozzles, several mask steps must be employed for creation of the final device including alignment of several patterning steps using photo masks. Alignment of features is often a critical parameter and the alignment accuracy is a function of the photolithography tool and the need for increased accuracy is often correlated to increased cost. For example, lithography utilizing a stepper tool offers much better pattern-to-pattern alignment than a contact aligner tool, however, at an instrument cost more than an order of magnitude higher. In addition stepper tools require a highly trained operator and maintenance costs are high. A process allowing use of a contact patterning tool allows a lower cost expensive for capital equipment, lower maintenance and operator cost. Additionally, the entire wafer patterning can be conducted with one single exposure opposed to needing multiple exposure by a stepper tool. Exposure cost for a contact alignment tool is independent of wafer size (except the cost of the mask creation itself) whereas a stepper tool cost increases with increasing diameter because of the increased number of exposures. Thus, there are several advantages to being able to use a contact alignment system.
The present invention is directed toward a method for improved lithography for patterning devices that require multiple mask alignment/overlays, for example, when fabricating microchip-based electrospray systems.
One aspect of the present invention is directed to a method for producing a nozzle of an electrospray device which includes providing a primary mask to accurately define the nozzle feature including the annulus and the through hole of the nozzle followed by the defining and etching of the primary mask containing the full nozzle feature. Then conducting a secondary mask step to repattern the through channel. The secondary mask serves to selectively mask given areas of the primary mask for subsequent etching. The through hole feature of the secondary mask aligns over the already patterned primary mask through channel, except that the secondary mask contains a slightly larger through channel diameter. This serves to mask off the annulus, but allowing the silicon through channel to be exposed for etching. The increased through channel size of the secondary mask feature is determined by the accuracy of the contact alignment tool.